Reactive Oxygen Species: Role in the Relaxation Induced by... : Journal of Cardiovascular Pharmacology (original) (raw)
The endothelium regulates coronary artery resistance by synthesizing and releasing both relaxing and contracting factors (1). Release of endothelium-derived relaxing factors can be stimulated by a variety of agents, including the vasoactive peptide bradykinin. It is well known that bradykinin elicits endothelium-dependent vasodilatation via prostaglandin I2 (PGI2) and/or NO (2,3). However, the vasodilator response to bradykinin can also be mediated in part by endothelium-derived hyperpolarizing factor (EDHF) (4). This diffusible factor produces vasodilatation and hyperpolarization of vascular smoothmuscle cells (VSMCs) (4-7). In the porcine coronary artery (PCA), phospholipase A2 and C are involved in the synthesis and/or release of EDHF (8); thus deesterification of a fatty acid derived from phospholipids (most probably arachidonic acid) must occur. Although cytochrome P-450 products (arachidonic acid metabolites) have recently been proposed as EDHF(s) in the bovine coronary artery (9) as well as the rat kidney and heart (10,11), in the PCA the nature of this factor remains unclear (12-14).
Associated with metabolism of arachidonic acid is the production of oxygen-derived free radicals (15,16). Endothelial cells can generate reactive oxygen species (ROS) on stimulation with bradykinin (17,18), and it has been shown that in cat (19) and mouse (20) cerebral arterioles the vasodilator effect of bradykinin or arachidonic acid is mediated by oxygen free radicals. There are several reports implicating superoxide anions, hydrogen peroxide (H2O2; not a free radical per se), or hydroxyl radicals in vasodilatation (19-23). Moreover, it has been shown that superoxide-mediated relaxation in the rabbit mesenteric artery involves a mechanism similar to that of the K+ channel opener cromakalim (21), and that H2O2 hyperpolarizes PCA VSMCs (23). Based on these studies, we hypothesized that an arachidonic acid metabolite or ROS, possibly formed via metabolism of arachidonic acid, may be involved in bradykinin- and/or arachidonic acid-induced relaxation via EDHF. To test this hypothesis, we examined (a) the role of arachidonic acid metabolism in the relaxation mediated by EDHF; (b) whether ROS can induce relaxation of PCA rings, as well as the mechanism(s) involved; and (c) the role of ROS in both bradykinin- and arachidonic acid-induced relaxation.
METHODS
Materials
Bradykinin (acetate salt), cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC), baicalein, eicosatetraynoic acid (ETYA), and 17-octadecynoic acid (17-ODYA) were obtained from Biomol (Plymouth Meeting, PA, U.S.A.); inorganic salts, prostaglandin F2α, N_ω-nitro-L-arginine methyl ester (L-NAME), arachidonic acid (sodium salt), indomethacin, clotrimazole, ouabain, cromakalim, catalase, superoxide dismutase (SOD), and xanthine/xanthine oxidase (X/XO) were purchased from Sigma (St. Louis, MO, U.S.A.). Indomethacin was dissolved in 0.2 M trisma base, baicalein and cromakalim in dimethyl sulfoxide (DMSO), CDC and clotrimazole in Na2CO3 (50 m_M), and ETYA and 17-ODYA in ethanol. All other chemicals were dissolved in distilled water. Ethanol or DMSO did not exceed 0.1% in the organ bath.
Isolated vascular-ring preparation
Pig hearts were obtained from a slaughterhouse and immediately immersed in ice-cold Krebs solution of the following composition (m_M_): NaCl (118.3); KCl (4.7); MgSO4·2H2O (1.2); KH2PO4 (1.2); CaCl2·6H2O (2.5); NaHCO3(25); and glucose (11.1). The left circumflex coronary artery was carefully removed and cleaned of fat and connective tissue. The arteries were cut into rings 3-5 mm wide, mounted on stainless steel stirrups (one fixed and the other movable), and suspended in an organ chamber (8 ml) filled with Krebs solution at 37°C, which was gassed continuously with 95% O2/5% CO2 to maintain a pH of 7.4. The movable stirrup was attached to an isometric force transducer (Grass, FT 03C, Quincy, MA, U.S.A.) coupled to a polygraph (Grass model 7D). The rings were stretched until basal tension reached 5 g and allowed to stabilize for 60 min. Rings were contracted with prostaglandin F2α (PGF2α; 1-10 μ_M_) or KCl (20-30 m_M_) to achieve a tension of ∼80% of the contraction obtained with 60 m_M_ KCl. Rings with and without endothelium were studied in parallel to test the role of the endothelial cells. The cells were removed by gently rubbing the internal surface of the vessel with a moistened cotton ball. Relaxation was expressed as the percent decrease in contraction produced by PGF2α or KCl.
Experimental protocols
Protocol 1: Role of arachidonic acid metabolism in the relaxation mediated by EDHF. In the presence of the cyclooxygenase inhibitor indomethacin (10 μ_M_), the vasodilator response to bradykinin (10−9-10−6_M_) in PGF2α- or KCl-contracted rings was tested in the presence or absence of the NO synthase inhibitor L-NAME (100 μ_M_) to confirm the presence of EDHF in our preparation. All of the following experiments were performed in the presence of indomethacin and L-NAME. Vasodilator responses to bradykinin (10−9-10−6_M_) and arachidonic acid (10−6-10−4_M_) were tested in PGF2α- or KCl-contracted rings with and without endothelial cells. The effect of the Na+/K+-ATPase inhibitor ouabain (5 × 10−7_M_) on vasodilatation was tested by adding it 60 min before contraction with PGF2α.
In another set of experiments, we studied whether arachidonic acid metabolism is involved in the vasodilatation mediated by EDHF. Coronary artery rings were pretreated with (a) ETYA (20 μ_M_) which inhibits all pathways of AA metabolism; (b) CDC (10 μ_M_) or baicalein (5,6,7-trihydroxyflavone; 1 μ_M_), which are both lipoxygenase inhibitors; or (c) clotrimazole (10 μ_M_) or 17-ODYA (2.5 μ_M_), which are both cytochrome P-450 inhibitors. All inhibitors were added 45 min before the rings were contracted. Finally, to determine whether preincubation with arachidonic acid potentiates bradykinin-induced relaxation, rings were exposed to arachidonic acid (100 μ_M_) for 45 min before the experiment. Nitroglycerin and cromakalim were used to test possible nonspecific effects of preincubation with arachidonic acid.
Protocol 2: Effect of ROS on vascular tone and their role in the relaxation induced by bradykinin and arachidonic acid via EDHF. The vasodilator response to the ROS-generating system xanthine (X; 100 μ_M_)/xanthine oxidase (XO; 0.02 U/ml) was tested in PGF2α- or KCl-contracted rings. Ouabain (5 × 10−7_M_) was added 60 min before the protocol. To determine which ROS was responsible for the vasodilatation induced by X/XO, rings were pretreated for 45 min with (a) SOD (500 U/ml), an enzyme that converts superoxide to hydrogen peroxide; (b) catalase (300 U/ml), an enzyme that converts hydrogen peroxide to water and oxygen; or (c) SOD and catalase combined. To study the role of ROS in the vasodilatation induced by bradykinin and arachidonic acid, rings were pretreated with SOD (500 U/ml), catalase (300 U/ml), or SOD plus catalase for 45 min.
Statistical analysis
Results are expressed as mean ± SEM. Differences between treatments in the same group were analyzed using a paired Student's t test. Student's two-sample t test or a Wilcoxon two-sample rank-sum test was used to analyze differences between groups, depending on whether assumptions of normality were met. For multiple comparisons, adjusted α levels were used to determine the significance of each test to ensure an overall testing level of 0.05.
RESULTS
Role of arachidonic acid metabolism in the relaxation mediated by EDHF
In the presence of the cyclooxygenase inhibitor indomethacin (10 μ_M_), PGF2α- or KCl-contracted rings relaxed in response to bradykinin (10−9-10−6_M_) in a dose-dependent manner (Fig. 1). L-NAME (100 μ_M_) completely inhibited the vasodilatation induced by bradykinin in KCl- but not PGF2α-contracted rings (Figs. 1 and 2). Bradykinin failed to induce relaxation in rings without endothelium (Fig. 2). Ouabain (5 × 10−7_M_), a Na+/K+-adenosine triphosphatase (ATPase) inhibitor, abolished the vasodilator response to bradykinin (Fig. 2). In rings contracted with PGF2α and treated with indomethacin and L-NAME, arachidonic acid (10−6-10−4_M_) induced dose-dependent relaxation, which was greatly diminished in deendothelialized rings (Fig. 3). As with bradykinin, the vasodilator response to arachidonic acid was abolished by KCl or ouabain (Fig. 3).
Effect of bradykinin (10−9-10−6 M) on prostaglandin F2α (PGF2α)- or KCl-precontracted porcine coronary artery rings in the absence (a, c) or presence (b, d) of N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_). All experiments were carried out in the presence of indomethacin (10 μ_M_).
Effect of bradykinin (10−9-10−6 M) on prostaglandin F2α (PGF2α) contracted porcine coronary artery rings with (•) and without endothelium [E(−); ○] and rings either pretreated with the Na+/K+-ATPase inhibitor ouabain (5 × 10−7 M; □) or contracted with KCl (▪). Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present throughout the experiment. Results are expressed as mean ± SEM.
Effect of arachidonic acid (10−6-10−4 M) on prostaglandin F2α (PGF2α)-contracted porcine coronary artery rings with (•) and without endothelium [E(−); ○] and rings either pretreated with the Na+/K+-ATPase inhibitor ouabain (5 × 10−7 M; □) or contracted with KCl (▪). Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present throughout the experiment. Results are expressed as mean ± SEM (n = 4). *p < 0.01, control versus E (−).
ETYA (20 μ_M_), which inhibits all arachidonic acid pathways, or the cytochrome P-450 inhibitor clotrimazole (10 μ_M_) slightly inhibited the vasodilatation induced by bradykinin (Figs. 4 and 5d, respectively). However, pretreatment with CDC (10 μ_M_) or baicalein (1 μ_M_), two different lipoxygenase inhibitors (Fig. 5a and b), or 17-ODYA (2.5 μ_M_), another cytochrome P-450 inhibitor (Fig. 5c), had no effect. Although ETYA slightly affected the vasodilator response to arachidonic acid, neither cytochrome P-450 nor lipoxygenase inhibitors had any effect (Fig. 6).
Effect of eicosatetraynoic acid (ETYA; 20 μ_M_), which inhibits all arachidonic acid pathways, on bradykinin-induced relaxation of prostaglandin F2α (PGF2α)-contracted porcine coronary artery rings. Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present throughout the experiment. Results are expressed as mean ± SEM (n = 5). *p < 0.01, control versus ETYA.
Effect of the lipoxygenase inhibitors cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC; a; 10 μ_M_) and baicalein (b; 1 μ_M_) and the cytochrome P-450 inhibitors 17-ODYA (c; 2.5 μ_M_) and clotrimazole (d; 10 μ_M_) on bradykinin-induced relaxation of prostaglandin F2α (PGF2α)-contracted PCA rings. Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present throughout the experiment. Results are expressed as mean ± SEM (n = 4-6). *p < 0.01, control versus clotrimazole.
Effect of eicosatetraynoic acid (ETYA; 20 μ_M_) (a), which inhibits all arachidonic acid pathways, and the lipoxygenase inhibitor cinnamyl-3,4-dihydroxy-α-cyanocinnamate (CDC; ○; 10 μ_M_) and the cytochrome P-450 inhibitors clotrimazole (▪; 1 μ_M_) and 17-ODYA (□; 2.5 μ_M_) (b) on arachidonic acid-induced relaxation of prostaglandin F2α (PGF2α)-contracted PCA rings. Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present throughout the experiment. Results are expressed as mean ± SEM (n = 4). *p < 0.01, control versus ETYA.
Pretreatment of PCA rings with arachidonic acid inhibited bradykinin-induced relaxation by 50%, which was reversed after washout (Fig. 7a). However, preincubation with arachidonic acid had no effect on the relaxant response to nitroglycerin (Fig. 7b), an endothelium-independent vasodilator, or cromakalim (Fig. 7c), a K+ channel opener.
Effect of pretreatment with arachidonic acid (AA; 100 μ_M_) on the relaxation induced by bradykinin (a; 10−9-10−6 M), nitroglycerin (b; 10−9-10−6 M), or cromakalim (c; 0.1-0.3 μg/ml). Rings were incubated with arachidonic acid for 45 min. Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present throughout the experiment. Results are expressed as mean ± SEM (n = 4-6). *p < 0.001, control versus AA.
Effect of ROS on vascular tone and its role in the relaxation induced by bradykinin and arachidonic acid via EDHF
Xanthine oxidase (XO; 0.02 U/ml) induced transient relaxation (Fig. 8a). Tension was restored to its initial value and did not change with time (Fig. 8a). Xanthine alone (X; 100 μ_M_) had no effect on tension; however, when xanthine was added in the presence of XO, it elicited time-dependent relaxation of PCA rings precontracted with PGF2α in the presence of the cyclooxygenase inhibitor indomethacin (10 μ_M_) and the NO synthase inhibitor L-NAME (100 μ_M_) (Fig. 8a and b). Relaxation was not affected by removal of the endothelium (Fig. 8b). In rings contracted with high extracellular concentrations of KCl (30 m_M_) or pretreated with the Na+/K+-ATPase inhibitor ouabain (5 × 10−7_M_), X/XO failed to cause relaxation (Fig. 8b). X/XO-induced relaxation was completely blocked by pretreatment with catalase (300 U/ml) or catalase combined with SOD (500 U/ml), but not by SOD alone, which actually potentiated the effect (Fig. 8a and c). SOD, catalase, or a combination of SOD and catalase had no effect on the vasodilatation elicited by either bradykinin or arachidonic acid (Fig. 9). The vasoconstriction induced by PGF2α was not affected by any of the treatments (Table 1).
a: Representative traces of the effect of xanthine oxidase (0.02 U/ml) alone or combined with xanthine (100 μ_M_) in the presence of vehicle, superoxide dismutase (SOD), or catalase on prostaglandin F2α (PGF2α)-contracted porcine coronary artery rings. b: Effect of xanthine oxidase plus xanthine on PGF2α-contracted porcine coronary artery rings with (•) and without endothelium [E(−); ○], rings pretreated with the Na+/K+-ATPase inhibitor ouabain (□; 5 × 10−7 M) on rings contracted with KCl (▪). c: Effect of SOD (○; 500 U/ml); catalase (▪; 300 U/ml), or a combination of both (□) on the relaxation induced by X/XO. Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present in all experiments. Results are expressed as mean ± SEM (n = 4 or 5). #p < 0.01; *p < 0.005, control versus KCl, catalase, or SOD + catalase.
Effect of superoxide dismutase (SOD; ○; 500 U/ml), catalase (▪; 300 U/ml), or SOD plus catalase (□) on relaxation induced by bradykinin (10−9-10−6 M) or arachidonic acid (10−6-10−4 M). Indomethacin (10 μ_M_) and N ω-nitro-L-arginine methyl ester (L-NAME; 100 μ_M_) were present throughout the experiment. Results are expressed as mean ± SEM (n = 4-7).
Effect of SOD, catalase, or SOD plus catalase on vasoconstriction induced by PGF2α in porcine coronary artery rings
DISCUSSION
Our findings show that in isolated PCA rings, EDHF induces relaxation by activating the Na+/K+-ATPase pump via a mechanism independent of NO, arachidonic acid metabolism, or ROS.
Besides NO and PGI2, another mechanism known to induce vasodilatation is hyperpolarization. It is well accepted that in porcine coronary arteries, the relaxant effect of bradykinin and arachidonic acid is mediated in part by EDHF (4). As expected, we found that the vasodilator response to bradykinin and arachidonic acid via EDHF was completely blocked when PCA rings were exposed to depolarizing concentrations of KCl, indicating that this action may be mediated by changes in membrane potential. It has been proposed that the relaxation and hyperpolarization induced by EDHF may be mediated by activation of the Na+/K+-ATPase pump (14,24,25), which exchanges three intracellular Na+ ions for two extracellular K+ ions and causes the cell to become negatively charged (26). In our preparation, the vasodilator response to bradykinin and arachidonic acid via EDHF was completely blocked by the Na+/K+-ATPase inhibitor ouabain, suggesting involvement of the Na+/K+-ATPase pump. These results are in agreement with those of Graier et al. (14), who found that ouabain inhibited the L-_N_G nitroarginine/indomethacin-resistant relaxation induced by the Ca2+ ionophore A23187 in the PCA, whereas apamin, an inhibitor of Ca2+-activated K+ channels, did not. Moreover, it was also shown that in PCA rings the vasodilator response to bradykinin via EDHF was not affected by blockade of voltage-dependent, Ca2+-dependent, or ATP-sensitive K+ channels (27). All of these data taken together strongly suggest that in the PCA, EDHF induces relaxation and hyperpolarization via activation of the Na+/K+-ATPase pump.
In bovine coronary arteries, EDHF(s) have been identified as EETs (9); however, the nature of EDHF in other vascular preparations, including the PCA (12-14), remains unclear. We found that ETYA, which blocks all arachidonic acid pathways, slightly inhibited the relaxant response to bradykinin and arachidonic acid, whereas clotrimazole, a cytochrome P-450 inhibitor, affected only the response to bradykinin. Neither 17-ODYA (another cytochrome P-450 inhibitor) nor lipoxygenase inhibitors affected either bradykinin- or arachidonic acid-induced relaxation. Confirming the results obtained by Weintraub et al. (12) in PCA rings, and in contrast to those reported by Hecker et al. (13), our data indicate that a metabolite of arachidonic acid is not likely to mediate the vasodilatation induced by bradykinin in PCA rings. This discrepancy may be related to the inhibitor concentrations used in the different studies. For example, at high concentrations clotrimazole is known to have nonspecific effects such as inhibition of both voltage-dependent (28) and Ca2+-dependent K+ channels (29).
Because it has been shown that phospholipase A2 and C are involved in the mechanism by which EDHF is synthesized and/or released (8,30), deesterification of a fatty acid derived from phospholipids (most likely arachidonic acid) must occur. Metabolism of arachidonic acid generates ROS (31). Moreover, the endothelium has the capacity to produce ROS in response to a variety of stimuli, including bradykinin (15,17-20) and arachidonic acid (15,19). The fact that arachidonic acid and bradykinin can stimulate generation of ROS, which have been shown to have vasoactive properties (19-22), suggests that they could directly induce relaxation or at least mediate the production of a relaxing factor (i.e., an oxidation product of arachidonic acid).
Furthermore, we considered the possibility that ROS may contribute to the EDHF-dilating effect of bradykinin and/or arachidonic acid. Because XO generates ROS (32), we tested the effect of X/XO on vascular tone and found that it caused sustained and time-dependent relaxation in the presence of the cyclooxygenase inhibitor indomethacin and the NOS inhibitor L-NAME. Dilatation was not affected by removal of the endothelium, suggesting that X/XO has a direct effect on VSMCs independent of NO and prostacyclin. The X/XO system produces superoxide radicals that are converted to H2O2 either spontaneously or via SOD. This H2O2 can be converted to water and oxygen by the enzyme catalase. The relaxation elicited by X/XO was abolished by catalase, whereas SOD failed to inhibit vasodilatation, suggesting that H2O2 rather than superoxide is involved; in addition, it was abolished by KCl, so that the effects of H2O2 may be mediated by changes in membrane potential. In support of our findings, Beny and von der Weid (23) reported that H2O2 induced hyperpolarization of porcine VSMCs. X/XO failed to cause relaxation in the presence of ouabain, suggesting that X/XO causes vasodilatation and hyperpolarization of VSMCs via a mechanism that involves generation of H2O2 as well as stimulation of the Na+/K+-ATPase pump.
Finally, we found that SOD, catalase, or SOD combined with catalase had no effect on the relaxant response to either bradykinin or arachidonic acid. Thus ROS are not likely to be involved in the synthesis, release, and/or action of the still unidentified hyperpolarizing factor that mediates the relaxant effect of bradykinin in isolated PCA rings.
In conclusion, EDHF induces relaxation of isolated PCA rings by activating the Na+/K+-ATPase pump via a mechanism independent of NO, arachidonic acid metabolism, or ROS. The identity of the EDHF in the PCA remains to be demonstrated.
Acknowledgment: This research was sponsored in part by NIH grants HL28982 and HL18575, and in part by a grant from the American Heart Association of Michigan to S. Pomposiello (13F956).
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Keywords:
Endothelium-derived hyperpolarizing factor; Reactive oxygen species; Na+/K+-ATPase pump; Bradykinin; Cytochrome P-450; Coronary artery
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